More than 7% of the US population and 20% of our veterans who have diabetes are not only at a 2 to 6-fold higher risk for having acute ischemic stroke but also suffer from unfavorable stroke outcome and poor recovery. Reperfusion therapy with tissue plasminogen activator (tPA) is the only therapy for ischemic stroke; however, this treatment increases the risk of bleeding into the brain (hemorrhagic transformation, HT) of diabetics. A critical barrier to progress in the development of new therapeutic strategies in this high-risk population is the lack of understanding how bleeding severity impacts stroke outcome & recovery in diabetes. The specific objective of this renewal application is to address this critical barrier by defining the impact and mechanisms by which HT impairs neurovascular repair after ischemic stroke in diabetes. During the past funding period we reported that a) there is robust pathological neovascularization of the brain in type 2 diabetes, b) a reperfusion injury superimposed on this pathology amplifies HT and worsens neurological deficits without increasing infarct size, and c) in the absence of reperfusion, there is no HT and no difference in functional outcome between control and diabetic animals. These novel results guided us to identify cerebral vasculature and HT as a therapeutic target in diabetic ischemic stroke. Danger-associated molecular patterns (DAMPs), intracellular molecules released upon cell death, is an emerging concept that is involved in the activation of the innate immune system via toll-like receptors (TLRs). Based on this foundation, the central hypothesis is that bleeding into the brain, petechial OR space-occupying, impairs neurovascular restoration and worsens outcome in diabetes via the activation of TLR-4 by excess iron, a novel DAMP. To achieve our overall goals, Aim 1 will test the hypothesis that even petechial nonspace-occupying HT impairs neurovascular restorative repair and worsens neurological deficits in diabetes. Aim 2 will test the hypothesis that iron deposition resulting from greater HT in diabetes impairs neurovascular plasticity and worsens outcome of ischemic stroke. Aim 3 will test the hypothesis that HT stimulates TLR4 signaling and inflammation worsening repair and recovery after diabetic ischemic stroke. The data obtained from our translational studies will yield the following outcomes: a) we will challenge the existing paradigm that only space-occupying HT worsens outcomes and demonstrate that any bleeding into the brain is detrimental by impairing vascular and neuronal repair, b) we will generate new and important data related to mechanisms of how diabetes attenuates neuronal and endothelial repair processes by using various combinations of animal models of diabetes or stroke to recapitulate the clinical condition, and c) we will identify iron as a new DAMP and show that iron chelation and/or downstream TLR4 inhibition are promising therapeutic targets in stroke treatment/recovery. This project will significantly impact stroke research, human health and VA mission because it will 1) identify neurovascular protection & restoration strategies to improve stroke outcomes, 2) advance our knowledge of the role of the cerebral vasculature in stroke repair, and 3) provide specific information on stroke recovery in diabetes which occurs in more than 30% of the 800,000 annual stroke victims. We are well poised to take this challenge since we have established collaborations with VA scientists that have provided support for the goals of this proposal.